IL-1β is produced by neutrophils and MDMs as a result of their transmigration across the BSCB. (A) Quantification of firm neutrophil adhesion at 10, 30, and 60 min after systemic infusions of fluorescently stained WT or IL-1β^−/− neutrophils into WT recipient EAE mice (n = 10–12). All data are expressed as means ± SEM with the mean representing the total number of firmly adherent neutrophils in eight fields of view (FOVs). (B) Scheme of the in vitro transmigration assay in which Gr1⁺ cells were allowed to transmigrate across a confluent monolayer of BMECs. (C) Flow cytometric analysis of DsRed expression in untransmigrated and transmigrated Gr1⁺ cells. (D and E) Quantification of the percentage of DsRed-expressing cells (D) and the mean DsRed fluorescence intensity (E) in untransmigrated versus transmigrated Gr1⁺ cells. Mean ± SEM of three independent experiments is shown. *, P < 0.05. (F and G) 2P-IVM of the spinal cord of LysM-GFP::pIl1b-DsRed double transgenic mice 2 d before onset (F) and at onset (G). Blood vessels were visualized by i.v. injection of a 10% solution of Qdot705 (blue). Myeloid cells (LysM-GFP⁺ cells) expressing the pro-form of IL-1β (DsRed signal) are shown with filled arrowheads. (H) Quantitative analysis of the DsRed fluorescence intensity (normalized to GFP fluorescence intensity) of circulating, rolling, adhering, and infiltrating (transmigrated) LysM⁺ cells (n = 3 mice/group). Means ± SEM are shown, and data are representative of two independent experiments.